Spore formation by Bacillus subtilis, an example of prokaryotic cellular differentiation, only occurs when cells are limited for essential nutrients. Such conditions lead to activation (phosphorylation) of SpoOA, a master regulatory protein responsible for negative and positive regulation of many genes expressed during stationary phase. Some of these genes are required for sporulation, but others are not; these latter genes are generally involved in adaptation to nutrient limitation, that is, in processes that are alternatives to spore formation. Fundamental questions about the earliest stages of sporulation remain to be answered. These include the nature of the nutritional or metabolic signals that initiate phosphorylation of SpoOA and control expression of genes induced early in stationary phase, the molecular pathway by which these signals influence SpoOA phosphorylation, and the mechanism by which the cell commits itself to sporulation as an alternative to slow growth in a nutritionally poor environment. This proposal seeks to identify the initial metabolic signal based on work in progress indicating that enzymes of the Krebs cycle must function in order for this signal to be effective. The notion that the Krebs cycle enzymes create a positive signal compound or eliminate a negative signal compound will be tested, as will the idea that Krebs cycle enzymes interact with proteins that participate in the SpoOA phosphorylation reactions. In previous work, a gene was identified whose product seems to interfere with synthesis or activity of SpoOA-phosphate. This may be a mechanism to delay the commitment to sporulation until cells have had a chance to attempt adaptation to slow growth conditions. This proposal seeks to test the idea that this protein (GsiAA) inhibits the pathway that phosphorylates SpoOA and seeks to identify the specific step sensitive to this inhibition. Many genes that are regulated by SpoOA-phosphate are also regulated by a second mechanism responsive to nutritional conditions. An example is the operon (dpp) responsible for transport of dipeptides. Mutations that relieve nutritional repression of dpp have been isolated and partially characterized. The direct repressor of dpp will be identified by genetic and biochemical experiments and will be tested for its activity at the dpp promoter and at other promoters subject to nutritional regulation. The metabolites that control the activity of the repressor will also be sought.